Bovine herpesvirus type 1 (BHV-1) up-regulates telomerase activity in MDBK cells

The proliferative capacity of mammalian cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumor progression and maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. Experimental data suggest that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. Evidence regarding the role of telomerase in the pathogenesis of several viruses including human immunodeficiency virus has led to an increased interest in the role of telomerase activity in other virus infections. In this research we evaluated the telomerase modulating activity of Bovine herpesvirus 1 (BHV-1) in MDBK cells. MDBK cells were infected at different multiplicity of infection with BHV-1 Cooper strain and telomerase activity at different times post-infection was measured by the TRAP assay. Our data indicate that BHV-1 significantly up-regulates telomerase activity at 3 and 6h post-infection decreasing after the 24h post-infection. Our data, showed that the effect was mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an immediate early gene is primarily responsible.     

A cohort study of telomere and telomerase biology in cats

OBJECTIVE: To investigate telomere lengths in tissues of domestic shorthair (DSH) cats of various ages, evaluate the relationship between telomere length and age of cats, and investigate telomerase activity in the somatic tissues of cats. SAMPLE POPULATION: Tissues obtained from 2 DSH cats and blood samples obtained from 30 DSH cats. PROCEDURE: DNA isolated from blood cells and somatic tissue samples was subjected to terminal restriction fragment (TRF) analysis to determine mean telomere repeat lengths. Protein samples were subjected to analysis by use of a telomeric repeat-amplification protocol to assess telomerase activity. RESULTS: MeanTRF values of cats ranged from 4.7 to 26.3 kilobase pairs, and there was significant telomeric attrition with increasing age of cat. Telomerase activity was not found in a wide range of normal tissues obtained from 2 cats. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results clearly indicates that telomeres are shorter in older cats, compared with young cats; therefore, telomeres are implicated in the aging process. The analysis of telomerase activity in normal somatic tissues of cats reveals a pattern of expression similar to that found in human tissues. IMPACT FOR HUMAN MEDICINE: Fundamental differences in the biological characteristics of telomeres and telomerase exist between humans and the other most widely studied species (ie, mice). The results reported here reveal similarities in telomere and telomerase biologic characteristics between DSH cats and humans. Hence, as well as developing our understanding of aging in cats, these data may be usefully extrapolated to aging in humans.     

Induction of telomerase activity in avian lymphoblastoid cell line transformed by Marek's disease virus, MDCC-MSB1

Telomerase has been studied extensively in human and murine tumors, but little is known about the role of telomerase in the tumor biology of other vertebrate species such as the chicken. We studied the telomerase activity of the lymphoblastoid cell line derived from lymphomas induced by Marek's disease virus (MDCC-MSB1) compared with another avian cell line (PA5) and peripheral blood lymphocytes (PBL) using the telomeric repeat amplification protocol (TRAP) Assay. Telomerase activity in MDCC-MSB1 was 4.5 times greater than in the PA5 cell line and normal avian lymphocytes. These results demonstrate for the first time that telomerase is more intense in one transformed cell line than in normal cells, suggesting a potential role for telomerase in carcinogenesis induced by an avian virus.     

Telomere length and telomerase activity in canine mammary gland tumors

OBJECTIVE: To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors. SAMPLE POPULATION: 27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs. PROCEDURE: Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay. RESULTS: Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean +/- SEM, 14.5+/-0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors.     

端粒和端粒酶与肿瘤关系的研究进展

端粒是末端染色体 ,维持染色体的稳定。端粒酶是染色体末端转移酶 ,是合成端粒DNA的酶 ,对端粒的结构起着稳定的作用。端粒、端粒酶的研究始于 2 0世纪初期 ,但直到近几年科研人员才发现他们与肿瘤有着密切的关系。在人类端粒酶的研究过程中发现端粒酶在约 85 %以上的恶性肿瘤中呈阳性 ,而动物肿瘤与端粒酶的研究还是空白。文章主要介绍端粒、端粒酶的结构功能及它们与肿瘤关系的研究进展 ,并讨论世界上首例蛋鸡 J亚群禽白血病的自然发病的病例的端粒酶 TERT表达。作者曾用辣根过氧化物酶标记的链酶卵白素进行免疫组化检测 ,结果在病鸡的心、肝、脾、肺、肾、气管、十二指肠、睾丸、骨髓、脑、胸腺、法氏囊等组织中发现 TERT表达呈阳性 ,说明在蛋鸡 J亚群禽白血病发生时端粒酶活性升高 ,提示端粒酶参与了该病的发生发展机制 ,并且该结果与人类肿瘤端粒酶的活性的研究结果相一致     

Telomerase activity in canine osteosarcoma

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25–40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase‐positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.     

端粒酶与肿瘤相关性研究进展

端粒是一段存在于真核细胞染色体末端,随着细胞分裂而缩短的特殊结构。端粒酶可延长端粒,但在正常人体细胞内活性较低或无活性。大多数肿瘤细胞通过激活端粒酶活性,延伸端粒达到细胞无限增殖的可能。端粒酶的活性与肿瘤的发生密切相关。本文以近年来临床常见的恶性肿瘤为出发点,综述了其与端粒酶活性关系及端粒酶抑制剂的最新研究。     

Telomerase activity as a marker for malignancy in feline tissues

OBJECTIVE: To establish the diagnostic significance of the telomeric repeat amplification protocol (TRAP) assay in detecting feline malignancies. SAMPLE POPULATION: Solid tissue specimens collected from 33 client-owned cats undergoing diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital between July 1997 and September 1999 and an additional 20 tissue samples were collected from 3 clinically normal control cats euthanatized at the conclusion of an unrelated study. PROCEDURE: The TRAP assay was used for detection of telomerase activity. Each result was compared to its respective histopathologic diagnosis. RESULTS: Twenty-nine of 31 malignant and 1 of 22 benign or normal tissue samples had telomerase activity, indicating 94% sensitivity and 95% specificity of the TRAP assay in our laboratory. CONCLUSIONS AND CLINICAL RELEVANCE: The diagnostic significance of telomerase activity has been demonstrated in humans and recently in dogs by our laboratory. We tested feline samples to determine whether similar patterns of telomerase activity exist. On the basis of our results, the TRAP assay may be clinically useful in providing a rapid diagnosis of malignancy in cats. The telomerase enzyme may also serve as a therapeutic target in feline tumors.     

端粒酶和细胞衰老

端粒是真核细胞染色体末端的一种特殊结构 ,由端粒 DNA和端粒蛋白质组成。端粒DNA是富含 G的高度保守的重复核苷酸序列 ,人和其他哺乳动物的端粒 DNA序列均由 5′-3′,方向 (TTAGGG) n 反复串联组成 ,它参与 DNA复制 ,对维持染色体的稳定和完全复制有重要作用。正常动物细胞 DNA的端粒随着细胞分裂而缩短 ,当缩短到一定长度时细胞将停止增殖并死亡。细胞中的端粒酶在正常细胞不表达 ,只在生殖细胞、干细胞和肿瘤细胞中表达。文章介绍了细胞衰老的细胞生物学机制、端粒酶和细胞衰老的关系 ,讨论了端粒酶和克隆动物的早衰现象的关系     

Abnormal structure of the canine oncogene, related to the human c-yes-1 oncogene, in canine mammary tumor tissue.

Cellular oncogenes of genomic DNA in 6 canine primary mammary tumors were screened by Southern blot analysis, using 7 oncogene probes. A canine genomic oncogene related to the human c-yes-1 oncogene was detected as abnormal bands in solid carcinoma genomic DNA digested with EcoRI, HindIII, HindIII-EcoRI, or HindIII-BamHI. Comparison was made between other tumor specimens and control specimens obtained from 4 clinically normal dogs--1 mixed breed and 3 Shiba Inu dogs (the same breed as the dog from which the solid carcinoma was obtained). These abnormal bands were 0.1 to 1 kilobase shorter than the normal gene. However, digestion of genomic DNA obtained from normal WBC of this dog also produced all of the abnormal bands as observed in digested DNA from the solid carcinoma tissue. Therefore, in this dog, the genomic DNA of all somatic cells from the ontogenic stage still had the abnormal sequences related to the human c-yes-1 oncogene, and it is possible that this abnormal structure may have some role (eg, as an initiator) in tumorigenesis or the progression of this tumor.     

Telomerase activity in clinically normal dogs and dogs with malignant lymphoma

OBJECTIVES: To determine whether telomerase activity was present in lymph nodes, buffy coat, and serum samples from dogs with malignant lymphoma (ML) and in liver, lymph node, buffy coat, and serum samples from clinically normal dogs SAMPLE POPULATION: Tissue specimens and blood samples were obtained from 11 clinically normal adult dogs (age range, 1 to 4 years) and 14 client-owned dogs with ML. PROCEDURE: The telomere repeat amplification protocol assay was used to quantify telomerase activity in the tissues from clinically normal dogs and dogs with ML. RESULTS: Of 11 clinically normal dogs, 8 had lymph node samples, 5 had liver samples, and 1 had buffy coat samples with detectable telomerase activity. None of the serum samples from the clinically normal dogs had detectable telomerase activity. Of 14 dogs with ML, 9 had lymph node samples, 3 had buffy coat samples, and 1 had serum samples with measurable telomerase activity. CONCLUSIONS AND CLINICAL RELEVANCE: Telomerase activity was not specific to tumor cells and overlapped with that found in cells from clinically normal dogs. Telomerase activity in neoplastic lymph nodes was not substantially different from that found in lymph nodes from clinically normal dogs. The determination of telomerase activity cannot be used as a sole diagnostic test for cancer. Therapeutic modalities directed toward the telomerase enzyme may not be feasible in dogs, because somatic tissues from clinically normal dogs possess variable amounts of telomerase activity.     

端粒和端粒酶与衰老关系的研究进展

端粒是存在于线性染色体末端的一段特殊的DNA－蛋白质复合物，由于末端不能复制，正常体细胞随着细胞分裂的进行而逐渐丢失端粒序列，导致细胞老化和死亡。端粒酶是维持端粒长度的一种特殊的DNA聚合酶，在细胞的增殖、衰老及永生中起重要作用。本文就端粒和端粒酶及其与衰老的作一综述。     

Telomerase: a potential diagnostic and therapeutic tool in canine oncology

In recent years there has been considerable interest in telomerase as a target for therapeutic intervention in oncology. This largely stems from the vast number of studies that have demonstrated expression and activity of the enzyme telomerase in the majority of human cancer tissues with little or no activity detectable in normal somatic tissues. These studies have led to an interest in the role of telomerase in cancers associated with domesticated species, in particular tumors that affect dogs. This article reviews the biology of telomerase and the biological significance of telomerase activity in canine tumors and discusses the clinical implications of telomerase expression in canine cancers with regard to therapeutics and diagnostics.     

Inhibition of telomerase in canine cancer cells following telomestatin treatment

Telomere shortening in normal somatic cells has been proposed as a major barrier to unlimited cellular proliferation. Telomerase is an enzyme capable of maintaining telomere length, and thus bypassing this barrier. In human beings, telomerase activity is restricted to cancer cells and cells of stem or germ cell lineages. Dogs represent a potentially useful clinical model for the development of telomerase‐based therapies because telomerase activity is also restricted to cancer cells and stem cells in this species. We examined the ability of telomestatin to inhibit telomerase activity in telomerase‐positive D17 and CMT7 canine cancer cell lines. At a concentration of 2 μM, telomestatin treatment resulted in a decrease in telomerase activity, telomere shortening, growth inhibition and apoptosis in telomerase‐positive cancer cells. These effects were not seen in telomerase‐negative skin fibroblasts or negative controls. These results confirm that telomestatin specifically inhibits telomerase activity in canine cancer cells and strengthens the usefulness of dogs as a model for testing telomerase‐based therapies.     

Measurement of telomerase activity in bovine leukaemia virus infected cows

Telomerase adds new telomeric sequences to the end of chromosomal DNA in order to overcome the end-replication problem. The upregulation of telomerase activity in tumours has been reported in humans and some mammals and is considered to be a tumour marker; however, such activity has not been investigated in cows. Therefore, we investigated telomerase activity in bovine leukaemia, the most common tumour in cows and its relationship with the bovine leukaemia virus (BLV) infection, which is the major cause of leukaemia. Telomerase activity was detected in 25 of 29 bovine leukaemia tissue samples. In peripheral blood lymphocytes (PBL) from BLV-infected cases that did not develop the tumour, telomerase activity was detected in 11 of 71 cases (15.5%). When these cases were classified based on serological tests and the peripheral blood lymphocyte count, the telomerase activity was observed to be the highest in the seropositive, non-lymphoproliferative (PBL<8000 microl(-1)) cases (three of seven cases, 42.9%), and not observed in the lymphoproliferative cases (PBL<16,000 microl(-1)) except in one case. Although the precise pathogenesis of BLV-related diseases remains obscure, persistent lymphocytosis is considered as a pre-neoplastic state. In contrast, our results suggested that given the fact that telomerase activity indicates tumour development, the aleukaemic stage could be defined as the 'pre-neoplastic state'. In conclusion, similar to many tumours in humans, telomerase activity was detected in bovine leukaemia; further, this activity can be a potentially useful prediction marker for tumour development and/or a good therapeutic target.     

端粒酶在细胞永生化中的应用

细胞永生化是目前细胞生物学研究的热点和难点之一。位于真核生物细胞染色体末端的端粒可以稳定染色体,而由RNA和蛋白质组成的端粒酶以自身RNA为模板合成端粒。因此,将外源性端粒酶逆转录酶（TERT）基因转染至目的细胞,则可能诱导细胞发生永生化。本文从端粒、端粒酶、端粒酶在细胞永生化中的应用、端粒酶在传代细胞系中的应用、存在问题与展望等五个方面进行了综述,以期为相关研究人员提供参考。     

Cellular proliferative and telomerase activity in canine mammary gland tumors

In canine mammary tumors, we examined the telomerase activity, proliferative activity by proliferative cell nuclear antigen (PCNA) immunohistochemistry, and percentage of apoptotic cells by the deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The relationship between these measures and histopathologic malignancy was also investigated. PCNA index was highest in malignant tumors (adenocarcinoma: 27.0%; malignant mixed tumor: 15.7%), followed by benign tumors (adenoma: 4.4%; benign mixed tumor: 5.3%), hyperplasia (2.1%), and normal mammary gland (0.9%). In adenoma and adenocarcinoma, papillary and solid types showing higher cellularity tended to have higher PCNA indices than did cystic and tubular types. Although the TUNEL index was <1% in all cases, the relationship between this measure and histopathologic diagnosis showed the same tendency as observed in PCNA immunostaining. Telomerase activity was detectable in all adenomas, benign mixed tumors, and adenocarcinomas examined. In contrast, all normal mammary glands, hyperplasias, and malignant mixed tumors were negative for telomerase. Relative telomerase activity (RTA) of adenocarcinoma (56.5) was significantly higher than that of adenoma (27.8) and benign mixed tumor (33.9), and a significant positive correlation (P < 0.001) was noted between RTA and PCNA index. No significant correlations were noted between either PCNA or TUNEL index and clinical features such as metastasis and tumor diameter. PCNA index and telomerase activity may be useful markers for judging malignancy of canine mammary tumors.